Raccoon poxvirus as a gene expression and vaccine vector for genes of rabies virus and other organisms

ABSTRACT

Raccoon poxvirus, an organism that may be indigenous in nature, is established as a suitable substrate for insertion and expression of nucleotide coding sequence of heterolgous organisms. Two infectious raccoon poxvirus recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants by thymidine kinase insertional inactivation. Raccoons that were fed polyurethane baits loaded with raccoon poxvirus recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with an otherwise lethal dose of raccoon rabies street virus. Dogs developed rabies virus neutralizing antibodies after feeding a vectored raccoon poxvirus recombinant in contrast to feeding a vaccinia: G recombinant that induced no rabies neutralizing antibodies. Cotton rats, skunks, mice and rabbits that were fed recombinant raccoon poxvirus developed variable levels of rabies neutralizing antibodies. All vaccinated cotton rats were protected from otherwise lethal rabies challenge.

This application is a continuation of application Ser. No. 07/198,213filed on May 25, 1988, now abandoned, which is a continuation in part ofco-pending U.S. patent application Ser. No. 07/010,424 filed Feb. 3,1987 and now abandoned.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention is related to rabies vaccines More particularly,the present invention is related to a rabies vaccine provided by theestablishment of a host organism as a substrate for inserting andexpressing genetic information of other organisms.

2. State of the Art

Raccoon pox virus (RCN) was first isolated from upper respiratorytissues of two apparently healthy raccoons captured in 1961-62. Theraccoons were captured during a survey of local wildlife at AberdeenProving Grounds, Maryland. This was reported by Herman, Y.F Bacteriol.Proc., 64th Ann. Meet. Amer. Soc. Microbiol. 1964 page 117, andAlexander. A.D. et al., J. Wildlife Dis. 1972:8:119-126. No grosslesions were seen during necropsy of the two raccoons. The sera of 22 of92 raccoons from the same area showed raccoon poxvirushemagglutinating-inhibiting (III) antibodies. This indicated thepresence of the virus in nature at that time. The current distributionof RCN virus in nature is unknown. The biological characteristics of oneisolate, including its DNA restriction map and its pathologicalinnocuity when inoculated into raccoons, has been described by ThomasE.K. et al., Arch. Virol. 1975:49:217-227; Esposito J.J. & Knight J.C.Virology 1985:143:230-251; and Parsons B.L. & Pickup D.J. Virology1987:161:45-53. In an earlier study by Esposito, J J. & Knight, J.C.Virology 1985:143:230-251 it was observed that thyymidine kinase (TK)nucleotide sequences within the DNA fragment Hind3-J of vaccinia viruscross hybridized with raccoon poxvirus DNA fragment Hind3-E. Chimericplasmids like those designed for inserting rabies virus surface spikeglycopiotein (G protein) coding sequences into the vaccinia virus TKregion were reported by Esposito et al., Virus Genes 1987:1:7-22 andEsposito, J.J. Brechling, K. Moss, B. Patent Application No. 07/010,424,filed on Mar. 25, 1987.

In the United States, the wildlife species most involved in rabiestransmission are skunks, raccoons, foxes, and insectivorous bats.Raccoons currently rank second to skunks as the major reservoir. Rabidfoxes are a major source of the disease in Canada and many Europeancountries. With the discontinued use of poisoning and trapping in the1960s, attempts have been made to protect foxes from rabies viabait-delivered oral vaccination. The immunization of foxes by thefeeding of live-attenuated rabies virus vaccine in sausages is disclosedby Baer, G.M. et al, Am. J Epidemiol. 1971:93:487-490 and thecorresponding U.S Pat. No. 4,014,991. This form of immunization led toefficacy and safety studies of live-attenuated rabies vaccine in chickenhead baits as described in the World Health Organization ExpertCommittee on Rabies 7th Report, 1984 Technical Report Series 709, WHO,Geneva. The virtual elimination of rabies in Switzerland and fieldtrials in West Germany and other countries have indicated that oral-baitrabies vaccines can significantly curtail the spread of rabies. Althoughlive-attenuated rabies virus vaccines are effective in immunizing foxesagainst rabies, they are not effective for immunizing skunks andraccoons.

A live recombinant vaccinia virus has been developed that expressed theG protein of rabies virus strain ERA and that induced rabiesvirus-neutralizing antibodies and protection against a rabies challenge.This is disclosed in the articles Wiktor, T.J. et al., PNAS1984:81:7194-7198 and Kieny, M.P. et al., Nature 1984:312:163-166. TheERA-G recombinant vaccinia virus has been inoculated to vaccinatevarious animals against rabies, including bovines, or by feeding therecombinant to foxes and raccoons. The possibility that a recombinantvaccinia virus might be introduced into nature, however, has raisedcontroversy largely because of the rare side effects in people that areassociated with primary smallpox vaccination with vaccinia virus.Furthermore, the vaccinia: ERA-G recombinant uses vaccinia virusCopenhagen, a strain that has been associated with relatively increasedside effect rates in humans when used during mass vaccinations againstsmallpox and increased virulence for laboratory animals as described byE. Krag-Anderson in Proceedings of Sypmposium on Smallpox 1969, pp.53-64, B. Gusic, editor, Yugoslave Academy of Sciences and Arts, Zagreb,1969 and by M.F. Polak in Symposium Series in ImmunobiologicalStandardization: R.H. 1973:19:235-242 Regamy and H. Cohen, editors, S.Karger Publishing, Basel.

In order to overcome problems associated with use of relatively invasivestrains of vaccinia virus like strain Copenhagen, six different chimericplasmids for regulating different levels of expression of G protein ofthe CVS strain of rabies virus have been produced and disclosed byEsposito et al. Virus Genes 1987:1:7-22. These plasmids are forinserting nucleotide coding sequences of G protein into the viral TKlocus of the New York Board of Health (NYBH) strain of vaccinia virus.This strain demonstrated relatively low side effect rates in peopleduring mass vaccination programs. In the six different recombinants thatwere produced, vaccinia virus promoter P₇.5 which is an early/late classpromoter or P₁₁ which is a late class promoter were used to driveexpression of G protein. As described by Esposito et al., Virus Genes1987:1:7-22, each of the six recombinants administered by intradermalscarification or by footpad injection protected mice against rabieschallenge. One recombinant that was injected intramuscularly into fivedogs provided protection against a lethal rabies challenge.

The industry is lacking a raccoon poxvirus as a substrate for insertinggenes to provide a useful expression system. The known systems do notprovide a recombinant raccoon poxvirus for expression of heterologousDNA such as rabies virus surface glycoprotein or recombinants such asraccoon poxvirus: rabies-G that can be produced by using chimericplasmids, such as those with viral thymidine kinase flanking sequencesand promoter sequences designed for production of vaccinia virusrecombinant. The industry further lacks live raccoon poxvirus: rabiesvirus recombinants that can be administered as an oral bait-deliveredvaccine to (1) protect raccoons and other wildlife species againstrabies and (2) produce related immune-reagents and/or other types ofveterinary vaccines.

SUMMARY OF THE INVENTION

The invention is a recombinant virus. The invention includes arecombinant raccoon poxvirus as a host organism. The raccoon poxvirushas a nucleotide coding sequence of a second organism. The nucleotidecoding sequence is desirably a segment of a rabies virus nucleotidecoding sequence.

The invention includes a pharmaceutical composition such as vaccinederived from the recombinant virus.

The invention includes a method for inducing protective immunity againstrabies and a kit for detecting the presence of rabies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a resolution of thymidine kinase nucleotide codingsequences within standard raccoon poxvirus and raccoon poxvirusrecombinants, and of rabies virus G protein encoding nucleotidesequences within recombinants.

FIG. 2 A, B, C depicts synthesis of G protein forms GI and GII asdetected by radioimmunoprecipitation assay of raccoon poxvirusrecombinant-infected cell cultures.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a recombinant raccoon poxvirus. Such recombinantviruses can be used for the production of oral or injectable vaccinesfor wildlife, livestock, or companion animals to control rabies and forthe production of related biochemical and immunobiological reagents.Raccoon poxvirus and vaccinia virus belong to same genus ofOrthopoxvirus and have genetic homology. For this reason, raccoonpoxvirus derived chimeric plasmids and many chimeric plasmids forinserting genes into vaccinia virus can be used interchangeably toproduce vectored recombinants. Raccoon poxvirus has unique biologicalproperties and appears to be indigenous in nature. This allows raccoonpoxviruses to have uses and applications similar to other live vectoredpoxviruses including vaccinia, a smallpox vaccine virus that is notindigenous in nature.

The invention is described by reference to two desirable embodiment,recombinant virus RCN-KB3-JE-13 (RAB-G? or RCN-GS62-JE-I. TheRCN-KB3-JE-13 (RAB-G) embodiment represents the preferred embodiment ofthe invention. This embodiment of the invention has been deposited intothe American Type Culture Collection, Rockville, Maryland underAccession Number VR2212 on May 23, 1988. Upon request, the Commissionerof Patents shall have access to the deposit which shall be viablymaintained for a period of 30 years or 5 years after the last request,or the life of the patent, whichever is longer. And upon issuance of apatent, the recombinants shall be made available to the public inaccordance with the law and with appropriate restrictions and guidelinesapplicable to the safe handling and use of this class of infectiousvirus.

For the purpose of this invention, the following definitions areprovided. A late promoter is defined as a DNA segment that includesnucleotide sequences that. regulate gene expression and are functionallyactive after, but not before, the replication of virus DNA in infectedcells. An early/late promoter is defined as a DNA segment that includesnucleotide sequences that regulate gene expression and are functionallyactive before and after the replication of virus DNA. This temporalactivity can be contiguous on or separable into subsets of thenucleotide sequences. Examples of late genes and early/late genes aredescribed by Rosel et al., J. Virol. 1986:60:436-439, Weir & Moss J.Virol. 1987:61:75-80, and Cochran et al., J.Virol. 1985:54:30-37.Generally such promoters reside immediately upstream of nucleotidecoding sequences for viral proteins. The above three documents and thedocuments cited below are hereby incorporated by reference.

The construction of a recombinant virus according to this inventioninvolves selecting a strain of raccoon poxvirus. An acceptable raccoonpoxvirus can include one of the two virus isolates from Aberdeen ProvingGrounds, Maryland as disclosed by Herman, Y.F. Bacteriol. Proc., 64thAnn. Meet. Amer. Soc. Microbiol., 1964, page 117; Alexander, A.D. etal., J. wildlife Dis. 1972:8:119:126; Thomas E.K. et al., Arch Virol.1975:49:217-227; Esposito J.J. & Knight J.C. Virology 1985:143:230-251;and Parsons B.L. & Pickup D.J. Virology 1987:161:45-53. The preferredembodiment of the invention utilizes thrice plaque-purified in CV1cells, RCN virus isolate designated as V71-I-85A by the Centers forDisease Control (CDC), Poxvirus Laboratory, Atlanta, Georgia 30333. TheDNA restriction electropherogram profile and map of this isolate hasbeen described by Esposito J.J. & Knight J.C. Virology 1985:143:230-251and Parsons B.L. Pickup D.J. Virology 1987:161:45-53. Another desirableisolate is designated V71-I-84 at the CDC and is also designated as ATCCVR-838 in the general repository catalog of The American Type CultureCollection, Rockville, Maryland 20852. This isolate has a similar, butnot identical, DNA restriction endonuclease electropherogram profilecompared to V71-I-85A and is characterized biologically by Thomas E.K.et al., Arch. Virol. 1975:49:217-227.

Insertion into the genome of RCN virus of nucleotide coding sequencesfor G protein is done by TK gene insertional inactivation using standardDNA recombination methodology that is well known in the art anddescribed by Mackett et al., In "DNA Cloning: A Practical Approach" D.M.Glover, Ed., 1985, 2:191-211, IRL Press, Washington, D.C. Moreparticularly, the methods described below for producing RCN recombinantswith the chimeric plasmids pGS62-JE-I (early/late promoter type), andthe pKB3-JE-13 (late promoter type) are essentially the same as thosefor producing NYBH vaccinia virus recombinants as disclosed by Espositoet al., Virus Genes 1987:1:7-22.

FIG. 1 shows resolution of TK coding sequences within RCN virus andrecombinants, and detection of insertion therein in recombinants ofrabies virus nucleotide coding sequences for G protein. Recombinant RCNviruses were produced by TK gene insertional inactivation using the samemarker rescue and recombinant selection methods detailed before withNYBH vaccinia virus and the described chimeric plasmids pGS62-JE-I(early/late promoter type) and pKB3-JE-13 (late promoter type). In FIG.1 Hind3 cleavage fragments are shown of virion DNA from thriceplaque-purified RCN virus (lanes 1, 4, 7), from recombinant virusRCN-GS62-JE-I (lanes 2, 5, 8), and from recombinant virus RCN-KB3-JE-13(lanes 3, 6, 9) resolved by agarose gel electrophoresis (lanes 1-3),then blotted bidirectionally onto nitrocellulose sheets. One sheet(lanes 4-6) was probed by hybridization with a NYBH vaccinia virus TKsegment, a Sau3A 0.888 kbp DNA fragment. The other sheet (lanes 7-9) wasprobed with G-cDNA. Migration positions of Lambda phage molecular weightmarker DNA Hind3 fragments are also shown.

The ability to select in culture medium containing bromodeoxyuridine forTK-inactivated vectored recombinants and the hybridizations in FIG. 1demonstrate that the location of inserted rabies virus G cDNA codingsequences is within the TK homologous sequence region of RCN virus.Comparison of electropherograms (lanes 1-3) of endonuclease cleaved DNAof standard RCN virus and recombinant RCN viruses, designated herein asRCN-GS62-JE-I and RCN-KB3-JE-13, show the DNA molecular weight increasethat took place in RCN virus DNA fragment Hind3-E (13.5 kilobase pairs(kbp)). After recombination with G-cDNA, a new fragment appeared,Hind3-D in the recombinants (RCN-GS62-JE-I fragment Hind3-D=15.45 kbp;RCN-KB3-JE-13 fragment Hind3-D=15.35 kbp). Autoradiograms of the threevirus DNAs (lanes 4-6) hybridized with the vaccinia virus TK cDNAresolve a single fragment of each virus, the aforementioned DNAfragments. Rabies virus G-cDNA, however, hybridized only with theHind3-D fragment of the two recombinant viruses.

FIG. 2 shows synthesis of G protein forms GI and GII detected byradioimmunoprecipitation assay of RCN recombinant virus-infected cellcultures. At 8 hours' postinfection (hpi) for RCN virus (panel C) or atthe times indicated for recombinants RCN-GS62-JE-I (panel B) andRCN-KB3-JE-13 (panel C), CV1 monkey kidney cell monolayers (multiplicityof infection=30 plaque-forming units (PFUs) purified virus per cell)were washed thrice with methionine-free culture medium, thenpulse-labelled for 15 minutes at 370° C. with medium that contained ³⁵S-methionine (100 uCi/ml). CVS rabies virus-infected (10 PFUs/cell) ormock-infected mouse neuroblastoma (Nb) cell monolayers (panel A) at 20hpi were pulse-labelled similarly for 4 hours. Clarified cell extractswere incubated first with rabbit antiserum prepared against detergent(NP-40) extracted CVS rabies virus G protein (antiserum shows somenucleoprotein (N) reactivity) and then with protein-A (S. aureus fixedbacteria). Proteins were eluted from the bacteria and resolved onSDS-polyacrylamide gels (12%) and autoradiographed. Migration ofmolecular weight marker proteins (K represents M_(r) =1000) are alsoshown.

The radioimmunoprecipitations revealed that forms GI (67 kilodaltons(kDa)) and GII (62 kDa) that are found in CVS rabies virus infectedcells comigrated with GI and GII forms that are expressed in cellmonolayers separately infected with the RCN recombinant viruses.Moreover, the time that G protein first appears in recombinantvirus-infected cells correlated with the expected time of activity ofvaccinia virus promoter P₇.5, 1 to 2 hours' postinfection (hpi), and ofP₁₁, 4 to 5 hours hpi. Further, an estimated tenfold higherconcentration of G protein was detected in cells infected withRCN-KB3-JE-13 virus (P₁₁ driving G expression) compared with thoseinfected with RCN-GS62-JE-I virus. Both RCN recombinants apparentlydirected faithful expression of G protein.

It is clear from the results illustrated in FIG. 1 that RCN virusaccepts vaccinia expression cassettes that are flanked by vaccinia TKnucleotide sequences to target recombination. It is also noted thatrabies virus G protein expressed in cell cultures via RCN virus isauthentic because of its reactivity with virus specific antibody inradioimmunoprecipitation tests showing production of the two differentglycosylated conformational forms of GI and GII. The vaccinia promoterP₁₁ in the RCN recombinants regulated higher levels of G expression thanP₇.5. These promoters functioned apparently the same as in earliervaccinia virus: G recombinants.

Six different animal species were vaccinated according to the inventionvia the oral route. These species responded in varying degrees inproducing rabies virus neutralizing antibodies (Nt. Abs). The resultsare reported in Tables 1 through 3 in the Examples below. Raccoons(Procyon lotor), a major reservoir animal for rabies in the UnitedStates, were fully protected against rabies virus challenge, suggestingthat RCN recombinants can be used in the formulation of an oraldelivered preparation of vaccine, such as an oral bait that containsvectored virus. The induction of rabies virus Nt. Abs. in 5 of 5 dogsand 1 of 3 skunks via the oral route of vaccination indicates that RCNrecombinant vaccines appears to have unique and novel properties versussimilar vaccinia virus based vaccines which have not yet proven to be aseffective in these animal species. Protection of mice by footpadinoculation of either RCN recombinant suggests that injectable vaccinescomprising immunogenic amounts of recombinant virus of the presentinvention in a pharmaceutically acceptable carrier, such as nontoxicbuffers and the like, is now made possible. An earlier report indicatedthat RCN virus may be indigenous in nature, a unique property not knownto be shared by vaccinia virus, which is generally considered assmallpox vaccine virus or virus propagated in laboratories. Of course,further studies on the host range, relative pathogenicity, currentdistribution of RCN virus in nature, together with regulatory scrutinyof such recombinants should be made before actual field trials of suchvaccine. Diagnostic kits or other immune-reagents based on the currentinvention are also now made possible.

EXAMPLES

In the Examples below, the procedures to produce the recombinants andthe vaccines were as described above. As previously described byEsposito et al., Virus Genes 1987:1:7-21, each of 6 NYBH vacciniarabies-G recombinants induced rabies virus Nt. Abs. and protected micewhen given by intradermal scarification or by footpad injection. OneNYBH vaccinia: rabies-G recombinant preparation injected intramuscularlyinto 5 dogs protected them against rabies challenge. Furthermore, when 2groups of 10 each of outbred mice were separately syringe-fed either of2 of the 6 NYBH recombinant preparations, about 20% protection wasnoted; the mice survived challenge only when rabies virus Nt. Abs. werepresent in the serum. Moreover, feeding one recombinant NYBH viruspreparation to 5 dogs induced no Nt. Abs. and was not protective. Theefficacy of the present invention is demonstrated for each of the twoRCN rabies-G recombinants that were tested by injecting or feeding themto mice, raccoons, or other animals.

EXAMPLE 1

Six-week-old colony-bred white, International Cancer Research (ICR)strain mice were separately fed the RCN recombinants by syringe at adose of 30 ul containing 10⁸ or 10⁹ PFUs per mouse. The results of thisexample together with the results of administering recombinants byfootpad inoculation are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Protection Against Street Rabies of Mice Immunized with                       Raccoon poxvirus: Rabies virus Glycoprotein Recombinants                                     Route:                                                         Virus          PFU dose/mouse Mortality                                       ______________________________________                                        RCN-GS62-JE-I  Footpad:                                                                      10.sup.9       0/9                                                            10.sup.8       0/12                                                           Oral:                                                                         10.sup.9       5/11                                                           10.sup.8       9/12                                            RCN-KB3-JE-13  Footpad:                                                                      10.sup.9       0/9                                                            10.sup.8       0/12                                                           Oral:                                                                         10.sup.9       1/10                                                           10.sup.8       7/12                                            ______________________________________                                    

Note: Raccoon poxvirus (RCN) recombinants RCN-GS62-JE-I or RCN-KB3-Je-13at the indicated plaque-forming units (PEU per 30 ul) per mouse(colony-bred ICR mice) were separately syringe-fed or injected in onerear footpad. At 4 weeks post vaccination a few animals were tested forrabies virus neutralizing antibodies (Nt. Abs - International Units/mlof serum) by the Rapid Immunofluorescent Focus Inhibition Test (Smith,J.S. et al., WHQ Monogr. Ser. 1973:23:354-357). Then, one month aftervaccination, the animals were challenged by footpad inoculation of about1 LD₅₀ of Mexico dog street rabies virus. Rabies deaths 1 monthpostchallenge are shown.

Every attempt was made to deposit the vaccine dose directly by syringeinto the mouth, only 23 of 45 mice orally vaccinated were protectedagainst a challenge with street rabies virus. Two of 8 serum samplesdrawn before challenge of the mice showed Nt. Abs., which suggested thatthe 50% protection observed can be explained if inadvertant intranasalinfection of these outbred mice was the reason for induction of the Nt.Abs. Alternatively, direct ingestion may have failed to produce uniforminfection of the digestive tract or some degree of natural resistance tooral infection may exist in ICR mice. In contrast, 42 mice (100%)resisted street rabies virus challenge as shown in Table 1 aftervaccination via footpad inoculation with either of the RCN recombinantswhich is similar to reported results with NYBH vaccinia: rabies-Grecombinants. Prechallenge screening of sera from 9 of the 42 mice ofthis group showed that all 9 had developed high titers of rabies virusNt. Abs. Taken together, the 2 positive sera from oral vaccinated micein Table I and the 9 positive sera from the footpad vaccinated miceshowed high titers of rabies Nt. Abs., (range per ml serum=4.5-10International United (I.U.). No malaise, generalized dermal lesions,loss of appetite, or abnormal excretions were apparent during monitoringof mice vaccinated by either route of inoculation. The difficulty ofuniformly infecting mice by feeding may be an advantageous property ofRCN recombinants in a field trial of oral-bait vaccines, where potentialfor sustained transmission of RCN recombinants among and between animalspecies may be undesirable.

EXAMPLE 2

Initially, for this example, one raccoon was syringe-fed 10⁸.5 PFUs ofRCN-GS62-JE-I virus. At one week postvaccination the raccoon showedsignificant rabies virus Nt. Abs. of 3.5 I.U./ml serum, rapidly risingto 80 I.U./ml by 2 weeks, then gradually leveling at 20 I.U./ml by 6months. Groups of raccoons were then fed tallow-coated polyurethanesponge-baits (3 cm³) that contained (in phosphate-buffered saline with20% fetal bovine serum (PBS-20FBS)) about 10⁹ PFU of the RCN: rabies-Grecombinant viruses or of standard RCN virus. No apparent pathologicabnormalities of any kind were noted during daily monitoring of thevaccinated raccoons. High titers of rabies virus Nt. Abs. developed by 4weeks' postvaccination in the animals fed the recombinants, and all ofthe recombinant vaccinated raccoons survived challenge with a streetstrain of raccoon rabies virus. These results are in Table 2.

                  TABLE 2                                                         ______________________________________                                        Antibodies and Protection Against Rabies of Raccoons                          Immunized with Raccoon poxvirus: Rabies virus                                 Glycoprotein Recombinants                                                                                          Post-                                                       Rabies            challenge                                Vaccine    Animal  virus    HI antibodies                                                                          rabies                                   virus      No.     Nt. Abs. RCN  VAC   death                                  ______________________________________                                        RCN-GS62-JE-I                                                                            29      20.3      80  80    -                                                 30      50.0     160  80    -                                                 31      50.0      80  80    -                                                 32      >100      80  40    -                                                 33      >100     320  80    -                                      RCN-KB3-JE-13                                                                            24      45.0     160  160   -                                                 25      48.5     320  80    -                                                 26      >100     320  160   -                                                 27      42.9      80  20    -                                                 28      40.7     160  40    -                                      RCN        17      <5       640  160   +                                                 35      <5       160  320   -                                                 36      <5       320  80    +                                                 37      <5       320  160   +                                      ______________________________________                                    

Note: Raccoon poxvirus (RCN) recombinants RCN-GS62-JE-I, RCN-KB3-JE-13,and standard RCN were fed separately to colony bred raccoons. Onetallow-coated polyurethane sponge-bait (3 cm³) filled with 10⁹ PFUs ofvirus in PBS was placed in each cage after food was withheld from theanimals for 24 hr. Animals were tested for rabies virus neutralizingantibodies (Nt. Abs.=International Units/ml of serum) by the RapidImmunofluorescent Focus Inhibition Test (Smith, J.S. et al., WHO Monogr.Ser. 1973:23:354-357), and for RCN or vaccinia (VAC) virushemagglutination inhibiting (HI) antibodies (reciprocal end-pointdilution of serum) (Thomas, E.K. et al., Arch. Virol. 49:217-227).Animals showed no prevaccinal HI or Nt. Abs.; titers at 4 weeks'postvaccination are shown. Two months after vaccination the animals werechallenged by bilateral masseter inoculation of a previously determinedlethal dose (≧5 LD ₅₀) of street rabies virus that had been isolatedfrom a raccoon in Virginia. Rabies deaths by 6 months' postchallenge areshown.

The results of this example also demonstrate that all the raccoonsdeveloped similar hemagglutination-inhibiting (HI)antibody levels to aRCN virus hemagglutinin (HA) or vaccinia virus HA preparation (Table 2).These data suggested that the HI test may be useful for serosurveys todetermine current distribution of RCN virus in wildlife.

EXAMPLES 3 THROUGH 6

In further efficacy experiments on host-range, and pathogenetic andimmunologic responsiveness of various animal species, recombinantRCN-KB3-JE-13 virus was syringe-fed to cotton rats, rabbits, dogs, andstriped skunks. The results of these examples are in Table 3.

                  TABLE 3                                                         ______________________________________                                        Rabies virus-neutralizing antibodies in four animal species                   vaccinated by syringe-feeding raccoon poxvirus: rabies-G                      recombinant RCN-KB3-JE-13                                                            Experment 4                                                                            Experiment 5                                                                              Experiment 6                                      Experiment 3                                                                           Skunk          Rabbit      Cotton                                    Dog No.                                                                              Ab    No.     Ab   No.   Ab    Rat No.                                                                              Ab                               ______________________________________                                        327    1.4   85-1    0.0  1     0.0   1      >4.5                             90     0.4   85-4    0.0  2     >4.5  2      >4.5                             32     1.8   85-5    1.8  3     0.0   4      >4.5                             39     0.1                4     >4.5  5      >4.5                              4     0.4                5     >4.5  7      4.5                                                        6     0.9   8      >4.5                                                                   10     0.4                                                                    11     0.0                              Mean   0.8           0.6        >2.4         >3.4                             Titer:                                                                        ______________________________________                                    

Note: Rabies virus-neutralizing antibody (Ab) titers are InternationalUnits per ml serum. Serum samples were from blood drawn at 4 weeks'postvaccination except for cotton rats which were test-bled at twoweeks' postvaccination. Recombinant RCN-KB3-JE-13 virus was syringe-fedas follows: dogs received 2 ml containing 10⁸.3 PFUs, striped skunksreceived 0.5 ml containing 10⁷.6 PFUs, rabbits received 0.5 mlcontaining 10⁷.6 PFUs, cotton rats received 0.1 ml containing 10⁷.1PFUs.

At the doses used (Table 3) most of the cotton rats (2 weeks'postvaccination (wpv)) and rabbits (4 wpv) showed relatively rapidproduction of significant levels of rabies virus Nt. Abs. (titer rangefor 6 rabbits 0-4.5 I.U./ml serum, for 8 cotton rats=0-4.5 I.U./ml); 1of 3 skunks, each fed 5×10⁷.1 PFUs, showed moderate Nt. Abs. (1.8I.U./ml). In contrast to the lack of induction of Nt. Abs. when a NYBHvaccinia: G recombinant was fed to dogs, 2×10⁸.1 PFUs of RCN-KB3-JE-13fed to each of 5 dogs induced moderate titers in each animal within 4wpv (range=0.1-1.4 I.U./ml). Standard and recombinant RCN virusinfection appeared innocuous for the 6 animal species vaccinated todate. At 6 months post vaccination, all recombinant vaccinated cottonrats survivied an otherwise lethal challenge by footpad injection ofrabies street virus. More dose-response determinations and rabieschallenges of these and other animals species are in progress.

What is claimed is
 1. A recombinant virus having all of thecharacteristics of ATCC deposit number VR
 2212. 2. A vaccine for rabies,comprising:a pharmaceutical excipient; and a therapeutically effectiveamount of a recombinant raccoon poxvirus, said raccoon poxvirus being ahost organism that contains a nucleotide sequence encoding a rabiesvirus glycoprotein G gene expressed via a vaccinia virus promoter.
 3. Amethod for inducing protective immunity against rabiescomprising:administering to a host, said host being susceptible torabies, an immunogenic amount of a vaccine for rabies, said vaccinehaving a recombinant raccoon poxvirus, said raccoon poxvirus being ahost organism that contains a nucleotide sequence encoding a rabiesvirus glycoprotein G, wherein said rabies virus glycoprotein G isexpressed via a vaccinia virus promoter.
 4. The method of claim 3wherein said administering of said vaccine is by oral baiting.
 5. Arecombinant virus comprising:raccoon poxvirus host organism; anucleotide coding sequence for rabies virus glycoprotein; and a vacciniavirus promoter which drives expression of said nucleotide codingsequence.
 6. The virus of claim 5, wherein said promoter is a vaccinialate promoter.
 7. The virus of claim 6, wherein said promoter is a P₁₁or P₇.5 promoter.
 8. A method for making a vaccine comprising arecombinant raccoon poxvirus, which comprises:providing a raccoonpoxviral DNA having a vaccinia virus late promoter, and inserting intosaid raccoon poxviral DNA a nucleotide sequence which encodes a rabiesvirus glycoprotein G, wherein said nucleotide sequence is linked to saidvaccinia virus late promoter so as to be expressed under the control ofsaid promoter.
 9. The method of claim 8 wherein said nucleotide sequenceis inserted in the thymidine kinase gene of the raccoon pox viral DNA.10. The method of claim 8, wherein said late promoter is a P₁₁ or P₇.5promoter.